Summary/Abstract The ability of immune cells to traffic from the blood stream to the sites of inflammation in order to find infectious agents and transmit information to other immune cells is a hallmark of the innate immune response. Trafficking is governed by the leukocyte adhesion cascade, a well-characterized, step-wise sequence of events in which blood borne immune cells tether, roll, firmly arrest, migrate and then enter tissues to perform immune cell functions. Neutrophils are the ?first responders? and will traffic to the sites of inflammation immediately upon sensing the inflammatory insult. These events normally occur within post-capillary venules, where cells must overcome high shear rates to bind to and eventually transmigrate through the endothelial surface. We and others have identified an interesting phenomenon wherein certain cells of hematopoietic origin - notably T-cells and hematopoietic stem and progenitor cells (HSPCs) ? will crawl upstream, against the direction of flow, on surfaces that contain the ligand intercellular adhesion molecule-1 (ICAM-1). This upstream migration is mediated by the ?2 integrin, ?L?2, also known as Lymphocyte Function-associated Antigen-1 (LFA- 1) binding to ICAM-1. Originally it was reported that neutrophils are unable to crawl upstream on ICAM-1, even though neutrophils express LFA-1. Neutrophils express an additional receptor for binding ICAM-1, Mac-1, which is up expressed when neutrophils are activated. We hypothesized that neutrophils are unable to crawl upstream because Mac-1 dominates binding ICAM-1 in neutrophils, and that if we block or disable Mac-1, the phenotype for upstream migration would be recovered. Our preliminary results show that by blocking Mac-1, upstream migration of neutrophils is recovered, thus setting the premise for this application. In this application we will show that neutrophils can indeed crawl upstream by inhibiting interactions of Mac-1, using both differentiated HL-60 cells and primary neutrophils. Specifically, in Aim 1, we will Determine the conditions in which neutrophils can crawl against the direction of shear flow, using antibody blocking to determine which integrins inhibit upstream migration in differentiated HL-60 cells and primary human neutrophils, and confirm that blocking Mac-1 leads to upstream migration. Then, in Aim 2, we will analyzing the altered directional migration preferences of neutrophils deficient in Mac-1, using RNA interference to make HL-60 cell lines that are deficient in Mac-1 and LFA-1, and test their directional preference for migration on purified molecular surfaces, as well as on stimulated HUVECs. We will validate the results of this aim with mutants of primary neutrophils by transducing CD34+ Hematopoietic Stem cells with shRNA against Mac-1 during their cytokine induced differentiation into neutrophils in-vitro. These studies will ultimately identify the critical surface receptors responsible in either guiding or preventing the upstream migration of neutrophils. This information will hold therapeutic potential for improving patient outcomes by better guiding neutrophils to the sites of inflammation and resolving inflammatory ailments.